Composition and device for detecting leukocytes in urine

ABSTRACT

Thiazole esters are suitable for detecting the presence of leukocytes in urine. Such thiazole esters are suitable for use in compositions, diagnostic devices, and methods for detecting the presence of leukocytes. In one embodiment, a thiazole ester is of the formula:or a salt or solvated salt thereof, in whichA is an N-blocked amino acid residue or an N-blocked peptide chain, preferably N-blocked alanine or N-blocked polyalanine; andR1 is unsubstituted or substituted heteroaryl; alkenyl substituted with unsubstituted or substituted aryl; or alkenyl substituted with unsubstituted or substituted heteroaryl. For example, R1 may be thienyl, pyridyl, furyl, styryl, pyrrolyl, or indolyl. In still another embodiment, the thiazole ester includes unsubstituted or substituted fused hydrocarbyl rings as a substituent. In one embodiment, fused hydrocarbyl rings include naphthyl. In another embodiment, fused hydrocarbyl rings include anthryl.

This application claims priority of Provisional Application No.60/116,613 filed Jan. 21, 1999 and No. 60/143,383 filed Jul. 12, 1999.

FIELD OF THE INVENTION

This invention relates generally to compounds, compositions, devices,and methods useful for detecting the presence of leukocytes through theactivity of leukocyte esterases and proteinases in urine.

BACKGROUND OF THE INVENTION

The presence of leukocytes in human urine is associated with infectionor malfunction in the kidney or urinary tract. Accurate detection has asignificant meaning for the physiological treatment or diagnosis of thepatient.

A basic method to measure the number of leukocytes in urine bymicroscope has been widely available for some time. However, thedisadvantages of this method include the investment of time and money inobtaining and installing the appropriate instrumentation. Further, falsenegatives can be obtained when samples are allowed to sit too longbefore analysis.

Research has been directed to developing other methods, such asindicator assays, that are suitable for detecting leukocytes moreeasily, conveniently, and accurately. Typical indicator assays use aspecific chemical substance (e.g., a substrate) that is degraded by oneor more enzymes present in leukocytes to create a product suitable foreffectuating a visible color change.

One such leukocyte assay is disclosed in U.S. Pat. No. 3,087,794 andinvolves the use of peroxidase that is contained in a granularleukocyte. This assay includes a filter paper stained with hydrogenperoxide and o-tolidine, which shows a colored oxidative product whencontacted with leukocytes. Yet this assay is less than desirable becauseperoxidase can be dangerous and reductive materials in urine may makeactual application impractical.

Other methods designed to confirm the presence of esterase andproteinase in leukocytes have also been developed. For example, onemethod uses a colorless or pale-colored ester compound as a substratethat is degraded by esterase into a colorless acid moiety and analcoholic moiety. Then, under diazonium or oxidative reaction, thealcoholic moiety is converted to a second densely colored product. Thismethod was derived from a process in which enzymatic degradation of thesubstrate naphtol-AS-D chloroacetate produced chloroacetate andnaphtol-AS. Reaction of the naphtol-AS product with a diazonium saltresulted in the formation of a colored azo compound.

This assay allows for the determination of leukocyte concentration bythe naked eye. Yet the use of this assay is less than desirable becausethe diazonium salt may react with urobilinogen or bilirubin contained inurine. As a result, concentrations of leukocytes greater than 500cells/μl are often necessary to avoid a false-negative reading.

U.K. Patent No. 1,128,371 discloses other possible substrates, i.e.,colorless indoxyl or thioindoxylesters. These substrates are degradedinto indoxyl or thioindoxyl by esterase. Colored indigo or thioindigomay then be produced by reaction with oxygen in the air or by anoxidizer. Yet the use of an assay with these substrates is less thandesirable because this is not sensitive and fails to detect leukocytesat a concentration less than 10,000 cells/μl.

Similarly, U.S. Pat. No. 4,278,763 suggests indoxyl-type substrates bydisclosing a method of using indoxyl or thioindoxylamino acid ester as asubstrate.

Other assays using pyrrole derivatives as substrates have also beendisclosed. For example, U.S. Pat. No. 4,704,460 discloses use of anamino acid ester of a pyrrole derivative as a substrate. When thisderivative is degraded in the presence of diazonium salt, a change insample color to deep violet results. Moreover, the addition of anucleophilic alcohol, such as decanol, greatly facilitates reactionrate, and in turn, allows detection of leukocyte concentrations as lowas 10 cells/μl within 90 seconds. Yet this assay is less than desirablebecause the syntheses of these two substrates is very difficult.

The above-described assays are characterized by numerous disadvantages.Some disadvantages include the detection of false negatives, theinterference of urobilinogen or bilirubin, the lack of sensitivity, andthe requirement of less-than-desirable substrates. Thus, it would bedesirable to identify new compounds and methods of using the compoundsto detect the presence of leukocytes in urine.

SUMMARY OF THE INVENTION

The invention is directed to thiazole esters suitable for detectingleukocytes in urine, compositions containing thiazole esters, diagnosticdevices suitable for detecting leukocytes in urine, and methods of usingthe thiazole esters or compositions thereof for detecting leukocytes inurine.

In one aspect, this invention is directed to novel thiazole esters.

One thiazole ester according to this invention is of the formula:

or a salt or solvated salt thereof, in which

A is an N-blocked amino acid residue or an N-blocked peptide chain,preferably N-blocked alanine or N-blocked polyalanine; and

R₁ is unsubstituted or substituted heteroaryl; alkenyl substituted withunsubstituted or substituted aryl; or alkenyl substituted withunsubstituted or substituted heteroaryl. For example, R₁ may be thienyl,pyridyl, furyl, styryl, pyrrolyl, or indolyl.

Another thiazole ester according to this invention is of the formula:

or a salt or solvated salt thereof, in which

A is an N-blocked amino acid residue or an N-blocked peptide chain,preferably N-blocked alanine or N-blocked polyalanine; and

R₂ is unsubstituted or substituted fused hydrocarbyl rings in which atleast one ring is aromatic. For example, R₂ may be naphthyl or anthryl.

In another aspect, this invention is directed to compositions thatinclude a thiazole ester of formula I or II. Compositions of theinvention may also include a diazonium salt such as2-methoxy-4-morpholinobenzene diazonium chloride, zinc chloride doublesalt.

In still another aspect, this invention is directed to diagnosticdevices that include a compound or composition of the invention. Suchdiagnostic devices include a substrate having a thiazole ester offormula I or II deposited thereon.

The compounds, compositions, and diagnostic devices of the invention arealso suitable for use in methods for detecting leukocytes in urine.Methods for detecting leukocytes in urine include contacting a thiazoleester of the invention and a diazonium salt with a urine sample.

The compounds and compositions suitable for use in devices and methodsof the invention are typically pharmaceutically acceptable.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to detecting leukocytes in urine. The presenceof leukocytes is typically detected by detecting the presence of enzymesknown in the art as leukocyte esterases and leukocyte proteinases thatare present with leukocytes.

Compounds of the invention include thiazole esters that contain an esterfunctionality, which is hydrolyzed upon exposure to leukocytes in urine.The hydrolysis of the ester of a thiazole ester of the inventionprovides a useful diagnostic technique because thiazolyl can react witha diazonium salt to produce an azo dye.

The compounds according to this invention are thiazole esters that aresuitable for use in compositions, diagnostic devices, and methods thatcan be used to detect leukocytes in urine.

One thiazole ester suitable for use in compositions and methods of theinvention is of the formula:

or a salt or solvated salt thereof, in which A is an N-blocked aminoacid residue or an N-blocked peptide chain, preferably N-blocked alanineor N-blocked polyalanine (e.g., alanine-alanine,alanine-alanine-alanine, and the like), being blocked at the aminoterminus by protecting groups known in the art, such as, for example,benzyloxycarbonyl, t-butoxycarbonyl, and p-toluenesulfonyl; and R₁ isunsubstituted or substituted heteroaryl; alkenyl substituted withunsubstituted or substituted aryl; or alkenyl substituted withunsubstituted or substituted heteroaryl.

The term “heteroaryl” includes heterocyclic aromatic derivatives havingat least one heteroatom, such as, for example, nitrogen, oxygen,phosphorus, or sulfur, and includes, for example, furyl, pyrrolyl,thienyl, oxazolyl, pyridyl, imidazolyl, thiazolyl, isoxazolyl,pyrazolyl, isothiazolyl, and the like. The term “heteroaryl” alsoincludes fused rings in which at least one ring is aromatic, such as,for example, indolyl, purinyl, benzofuryl, benzothienyl, quinolyl,4,5,6,7-tetrahydro-1H-indolyl, and the like. In the case of fused ringsthat have a hydrocarbyl ring fused to a heterocyclic ring, such as, forexample, benzothienyl and indolyl, the fused rings may be bonded tothiazolyl through either the hydrocarbyl ring or the heterocyclic ring.In some embodiments, heteroaryl includes a 5-membered ring. In otherembodiments, heteroaryl includes a 6-membered ring.

Such heteroaryl groups may be unsubstituted or substituted on the ringby, for example, aryl, heteroaryl, alkyl, alkenyl, alkoxy, amino, acyl,halo, nitro, cyano, —SO₃H, or hydroxy, in which such substituents mayfurther be substituted by aryl, heteroaryl, alkyl, alkenyl, alkoxy,amino, acyl, halo, nitro, cyano, —SO₃H, or hydroxy. In some embodiments,heteroaryl is substituted with alkyl, such as methyl, ethyl, propyl, orbutyl. In other embodiments, heteroaryl is substituted with alkoxy, suchas methoxy, ethoxy, propoxy, or butoxy. In still other embodiments,heteroaryl is substituted with aryl or heteroaryl.

The term “aryl” includes aromatic hydrocarbyl, such as, for example,phenyl, including fused aromatic rings, such as, for example, naphthyl.

The term “alkyl” includes a straight or branched saturated aliphatichydrocarbon chain having from 1 to 4 carbon atoms, such as, for example,methyl, ethyl, propyl, isopropyl (1-methylethyl), butyl, t-butyl(1,1-dimethylethyl), and the like.

The term “alkenyl” includes an unsaturated aliphatic hydrocarbon chainhaving from 2 to 4 carbon atoms, such as, for example, ethenyl,1-propenyl, 2-propenyl, 1-butenyl, 2-methyl-1-propenyl, and the like.

The above alkyl or alkenyl groups may optionally be interrupted in thechain by a heteroatom, such as, for example, a nitrogen or oxygen atom,forming an alkylaminoalkyl or alkoxyalkyl group, for example,methylaminoethyl or methoxymethyl, and the like.

The term “alkoxy” includes alkyl as defined above joined to an oxygenatom having from 1 to 4 carbon atoms in a straight or branched chain,such as, for example, methoxy, ethoxy, propoxy, isopropoxy(1-methylethoxy), butoxy, t-butoxy (1,1-dimethylethoxy), and the like.

The term “amino” includes as substituent of the formula —N(R₃)₂ in whicheach R₃ is independently hydrogen or alkyl. The term “alkyl” is asdefined above.

The term “alkenyl substituted with unsubstituted or unsubstituted aryl”or “alkenyl substituted with unsubstituted or substituted heteroaryl”includes alkenyl as defined above, and such groups are substituted withunsubstituted or substituted aryl or unsubstituted or substitutedheteroatyl, respectively. The terms “aryl” and “heteroaryl” are asdefined above. Examples of suitable substituted alkenyls include styryl,cinnamyl, furylethenyl, pyridylethenyl, and thienylpropenyl.

In some embodiments, alkenyl substituted with unsubstituted orsubstituted aryl is alkenyl substituted with unsubstituted orsubstituted phenyl. This phenyl, in some embodiments, is substitutedwith alkloxy, such as methoxy, ethoxy, propoxy, or butoxy. In otherembodiments, alkenyl substituted with unsubstituted or substitutedheteroaryl is alkenyl substituted with a nitrogen-containing ring,oxygen-containing ring, or sulfur-containing ring.

Examples of R₁ groups for thiazole esters suitable for use incompositions and methods of the invention include 2-thienyl; 4-pyridyl;2-furyl; β-styryl; 1,2-dimethyl-4-pyrrolyl; and 3-indolyl. It should beappreciated that one skilled in the art, having read this specification,would understand that a heteroatom may be at any one of severalpositions in a ring.

Another thiazole ester suitable for use in compositions and methods ofthe invention is of the formula

or a salt or solvated salt thereof, in which A is an N-blocked aminoacid residue or an N-blocked peptide chain, preferably N-blocked alanineor N-blocked polyalanine (e.g., alanine-alanine,alanine-alanine-alanine, and the like), being blocked at the aminoterminus by protecting groups known in the art, such as, for example,benzyloxycarbonyl, t-butoxycarbonyl, and p-toluenesulfonyl; and R₂ isunsubstituted or substituted fused hydrocarbyl rings in which at leastone ring is aromatic.

Fused hydrocarbyl rings include at least two hydrocarbyl rings thatmaintain at least one bond in common between the rings, for example,naphthyl, anthryl, 5,6,7,8-tetrahydronaphthyl, phenanthrenyl,triphenylenyl, 1H-fluorenyl, and the like. Fused hydrocarbyl rings maybe substituted by, for example, aryl, heteroaryl, alkyl, alkenyl,alkoxy, amino, acyl, halo, nitro, cyano, —SO₃H, or hydroxy, in whichsuch substituents may further be substituted by aryl, heteroaryl, alkyl,alkenyl, alkoxy, amino, acyl, halo, nitro, cyano, —SO₃H, or hydroxy.These substituents are as defined above.

In some embodiments, fused hydrocarbyl rings contain at least one5-membered ring. In other embodiments, fused hydrocarbyl rings containat least one 6-membered ring. In still some embodiments, fusedhydrocarbyl rings are substituted on at least one ring with alkyl, suchas methyl, ethyl, propyl, or butyl. In other embodiments, fusedhydrocarbyl rings are substituted on at least one ring with alkoxy, suchas methoxy, ethoxy, propoxy, or butoxy.

In one embodiment, R₂ is 1-naphthyl.

A composition of the invention includes a thiazole ester of formula I orII. Such a composition may also include a diazonium salt such as2-methoxy-4-morpholinobenzene diazonium chloride, zinc chloride doublesalt.

Compositions of the invention may be free of salts known in the art tohave an accelerating action. Examples of salts that give an acceleratingaction include salts of monovalent and divalent cations of the alkalimetals and alkaline earth metals, such as, for example, Li⁺, Na⁺, K⁺,and Mg⁺⁺ as well as their typical anions.

A diagnostic device suitable or detecting leukocytes in urine includes asubstrate suitable for supporting a thiazole ester of the invention,such as, for example, filter paper, filtration membrane, and other inertcarriers. These substrates are known in the art. The diagnostic devicealso includes one or more thiazole esters of formula I or II. Thiscompound is included with the substrate by, for example, sedimenting thederivative onto the substrate.

Generally, a novel diagnostic device may be prepared by combining thethiazole ester, an accelerator, and a diazonium salt. Acceleratorssuitable for use in compositions and methods of the invention are knownin the art and include, for example, octanediol and decanol.

Diazonium salts suitable for use in compositions and methods of theinvention are known in the art and include compounds that may be usedfor color-developing technology, such as, for example,1-diazo-8-naphtol-3,6-disulfonic acid, chloride, zinc chloride doublesalt; 6-diazo-1naphtol-3-sulfonic acid, chloride double salt; and2-methoxy-4-morpholinobenzene diazonium chloride, zinc chloride doublesalt. Preferably, the diazonium salt used in accordance with theinvention has no interaction with urobilinogen and/or bilirubin.

Methods for manufacturing a device according to this invention are knownin the art. Generally, a first solution containing boric acid andpolyvinylpyrrolidone is deposited onto a substrate, such as a filterpaper. Then a second solution containing a thiazole ester of theinvention and a diazonium salt, such as 2-methoxy-4-morpholinobenzenediazonium chloride, zinc chloride double salt, is deposited onto thesubstrate. Both solutions deposited onto the substrate may be free of asalt known in the art to have an accelerating action as described above.

To prepare a thiazole ester of the invention, a thiazolone derivativeand an alanine or polyalanine derivative may be used as startingmaterials. Thiazolones may be synthesized by reacting carboxymethylthiobenzimidate hydrobromide and pyridine.

As illustrated in the following scheme, the synthesis of a thiazoleester of the invention includes the reaction of a thiazolone derivativeof the general formula (2), and an amino acid chloride of the generalformula (3).

Each starting material is dissolved in solvent, and the resultingsolutions are cooled and mixed slowly. This reaction mixture is stirredand then allowed to stand at room temperature for several hours. Thereaction mixture is then washed, dried, filtered, and concentrated underreduced pressure. The solid, so formed, is dissolved in acetone andhexane is added. To remove any resulting hemisolid impurity, thereaction mixture is allowed to stand at low temperature for about 1hour. Another portion of hexane is added to the residue and the mixtureis refrigerated for 10 hours. The desired product is then filtered anddried.

Thiazolone derivatives tend to be highly reactive, resulting indimerization or multimerization even during recrystallization. This maylead to poor yield. Thus, for the current invention, pure substances areisolated from the final product of the synthesis of the thiazole esterwithout purifying thiazolone of the general formula (2). This method ofsynthesis enhances the yield of the derivatives of the invention.

In contrast to this, the aforementioned indole or pyrrole derivativeshave some recognized disadvantages. Many side reactions occur in theprocess of manufacturing their intermediates. Moreover, the reactionmechanism is very complicated and the yield for indole and pyrrolederivatives proves to be poor. But the synthesis of the thiazole estersof this invention allows for larger amounts of final product to beproduced by a general method within a relatively short period of time.Moreover, because the method of synthesizing the thiazole esters of theinvention is relatively simple and general, mass-scale production ismore practical.

Methods of the invention include detecting the presence of leukocytes inurine. To detect the presence of leukocytes in urine, a urine sample iscontacted with a thiazole ester of the invention and a diazonium salt inthe presence of an accelerator. If leukocytes are present, then areaction between thiazolyl and the salt produces an azo dye having aviolet color.

Typically such a method is directed to contacting a urine sample with asubstrate, for example, filter paper, that has a first solution of boricacid and polyvinyl pyrrolidone deposited thereon and then a secondsolution of a thiazole ester of formula I or II; a diazonium salt; andan accelerator deposited thereon. This method may be carried out free ofan accelerating salt. When a urine sample containing leukocytes contactssuch a substrate, then a violet color, which appears on the substrate,is observed.

WORKING EXAMPLES

This invention will be further characterized by the following examples.These examples are not meant to limit the scope of the invention, whichhas been fully set forth in the foregoing description. Variations withinthe scope of the invention are apparent to those skilled in the art.

EXAMPLE 1 N-(p-Toluenesulfonyl)-alanine

To synthesize a starting material containing a blocked N-alanine,N-(p-toluenesulfonyl)-alanine is first synthesized.

50.0 g of p-toluenesulfonyl chloride was added to 100 ml of 90° C.toluene with stirring. The p-toluenesulfonyl chloride solution intoluene was slowly added to 25.0 g of L-alanine dissolved in 500 ml of1N NaOH cooled to 5° C. and stirred for 24 hours. The resulting aqueouslayer is separated and cooled to below 5° C. and with the addition ofconcentrated hydrochloric acid, pH was adjusted to 1.5. After standingin the refrigerator for 3 to 4 hours, the white solid crystals, soformed, were filtered, twice washed with water, and dried.

EXAMPLE 2 N-Tosyl-L-alanyl chloride

The blocked starting material is next produced by reactingN-(p-toluenesulfonyl)-alanine and oxalyl chloride.

30.0 g of N-(p-toluenesulfonyl)-alanine and 6 to 7 drops of DMF(dimethylformamide) were dissolved in 100 ml of dichloromethane and 5 mlof oxalyl chloride was added dropwise. After stirring for 3 hours atroom temperature the reaction mixture was evaporated down to a residuewhich was dissolved in 100 ml of dichloromethane. The dichloromethanewas again evaporated and the resulting residue dissolved in 50 ml ofdichloromethane followed by the addition of 100 ml hexane. Afterstanding in the refrigerator overnight the crystals, so formed, werefiltered and dried.

EXAMPLE 3 2-(4-Pyridyl)-4-thiazol

HCl gas is bubbled through a solution of 10 g of 4-cyanopyridine and 50g of mercaptoacetic acid in approximately 20 ml of ether and 30 ml ofchloroform for 24 hours. During the reaction 30 ml of ether and 50 ml ofchloroform are added. The crystallized product, so formed, is filtered,washed with a solvent system of 1:1 ether and chloroform, and dried.

EXAMPLE 4 2-(2-Thienyl)-4-thiazol

HCl gas was bubbled through a solution of 10 g of3-thiophenecarbonitrile and 35 g of mercaptoacetic acid in approximately50 ml of ether for 24 hours. The crystallized product, so formed, wasfiltered, washed with 200 ml ether, and dried.

EXAMPLE 5 2-(1-Naphthyl)-4-thiazol

HCl gas was bubbled through a solution of 25 g of 1-naphthonitrile and25 g of mercaptoacetic acid in approximately 150 ml chloroform for 24hours. The crystallized product, so formed, was filtered, washed with200 ml chloroform, and dried.

EXAMPLE 6 2-(1,2-Dimethyl-4-pyrrolyl)-4-thiazol

HCl gas was bubbled through a solution of 5 g of1,5-dimethyl-2-pyrrolecarbonitrile and 5 g of mercaptoacetic acid inapproximately 50 ml of ether/chloroform (1:1) for 24 hours. Thecrystallized product, so formed, was filtered, washed with 100 mlether/chloroform (1:1), and dried.

EXAMPLE 7 2-(3-Indolyl)-4-thiazol

HCl gas is bubbled through a solution of 5 g of 3-cyanoindole and 5 g ofmercaptoacetic acid in approximately 50 ml of ether/chloroform (1:1) for24 hours. The crystallized product, so formed, is filtered, washed with100 ml ether/chloroform (1:1), and dried.

EXAMPLE 8 2-(2-Furyl)-4-thiazol

HCl gas was bubbled through a solution of 5 g of 2-furonitrile and 5 gof mercaptoacetic acid in approximately 50 ml of ether/chloroform (1:1)for 24 hours. The crystallized product, so formed, was filtered, washedwith 100 ml ether/chloroform (1:1), and dried.

EXAMPLE 9 2-(β-Styryl)-4-thiazol

HCl gas was bubbled through a solution of 25 g of cinnamonitrile and 25g of mercaptoacetic acid in approximately 150 ml of ether/chloroform(1:1) for 24 hours. The crystallized product, so formed, was filtered,washed with 200 ml ether/chloroform (1:1), and dried.

EXAMPLE 10 2-(4-Pyridyl)-4-(N-tosyl-L-alanyloxy)thiazol

5.0 g of 2-(4-pyridyl)-4-thiazol is dissolved in a cosolvent of 100 mlof dichloromethane and 10 ml pyridine and cooled to 5° C. A solution of15.0 g of N-tosyl-L-alanyl chloride in 50 ml dichloromethane is thenadded dropwise to the above mixture. The reaction solution is stirred atroom temperature or until the reaction is complete. The solution iswashed with 1N hydrochloric acid, water, saturated sodium bicarbonate,and a saturated sodium chloride solution in that order; dried overmagnesium sulfonate; filtered; and concentrated under reduced pressure.The residue, so formed, is dissolved in 100 ml of acteone and 100 ml ofhexane is added. After standing in the refrigerator for 1 hour, darksemisolid impurities are decanted off and the remaining solventevaporated. The amorphous solid, so formed, is dissolved in a minimum ofethylacetate, the resulting solution is then applied to a silica columnand eluted with a 2:1 ethylacetate/hexane solvent system. Fractionscontaining the desired product are collected, the solvent is evaporated,and the resulting residue is dissolved in 20 ml of acetone followed bythe addition of 50 ml hexane. After standing in the refrigerator theproduct, so formed, is filtered and dried.

EXAMPLE 11 2-(2-Thienyl)-4-(N-tosyl-L-alanyloxy)thiazol

5.0 g of 2-(3-thienyl)-4-thiazol was dissolved in a cosolvent of 100 mlof dichloromethane and 10 ml pyridine and cooled to 5° C. A solution of15.0 g of N-tosyl-L-alanyl chloride in 50 ml dichloromethane was thenadded dropwise to the above mixture. The reaction solution was stirredat room temperature until the reaction was complete. The solution waswashed with 1N hydrochloric acid, water, saturated sodium bicarbonate,and a saturated sodium chloride solution in that order; dried overmagnesium sulfonate; filtered; and concentrated under reduced pressure.The residue, so formed, was dissolved in 100 ml of acteone and 100 ml ofhexane was added. After standing in the refrigerator for 1 hour, darksemisolid impurities were decanted off and the remaining solventevaporated. The amorphous solid, so formed, was dissolved in a minimumof ethylacetate, the resulting solution applied to a silica column andeluted with a 2:1 ethylacetate/hexane solvent system. Fractionscontaining the desired product were collected, the solvent wasevaporated, and the resulting residue was dissolved in 20 ml of acetonefollowed by the addition of 50 ml hexane. After standing in therefrigerator the product, so formed, was filtered and dried.

The product was identified by a proton NMR analysis. A sample of theproduct was dissolved in acetone-d₆ (19 mg/0.7 ml) and a proton spectrumwas collected following AMRI SOP INS-041 using a Bruker® 300 MHz NMRSpectrometer (Bruker-Physik AG, Germany).

The identity of the product was further confirmed by infrared spectralanalysis by first mixing 263 mg of potassium bromide with 1.7 mg of theproduct and then grinding them in a Wig L Bug® (Crescent DentalManufacturing Co., Illinois). The ground mixture was pressed into adish, and an infrared spectrum was collected by a Spectrum 1000 IRSpectrometer following AMRI SOP INS-026. The sample was scanned between4000 and 400 cm⁻.

EXAMPLE 12 2-(1-Naphthyl)-4-(N-tosyl-L-alanyloxy)thiazol

5.0 g of 2-(1-naphthyl)-4-thiazol is dissolved in a cosolvent of 100 mlof dichloromethane and 10 ml pyridine and cooled to 5° C. A solution of15.0 g of N-tosyl-L-alanyl chloride in 50 ml dichloromethane is thenadded dropwise to the above mixture. The reaction solution is stirred atroom temperature until the reaction is complete. The solution is washedwith 1N hydrochloric acid, water, saturated sodium bicarbonate, and asaturated sodium chloride solution in that order; dried over magnesiumsulfonate; filtered; and concentrated under reduced pressure. Theresidue, so formed, is dissolved in 100 ml of acteone and 100 ml ofhexane is added. After standing in the refrigerator for 1 hour, darksemisolid impurities are decanted off and the remaining solventevaporated. The amorphous solid, so formed, is dissolved in a minimum ofethylacetate, the resulting solution is then applied to a silica columnand eluted with a 2:1 ethylacetate/hexane solvent system. Fractionscontaining the desired product are collected, the solvent is evaporated,and the resulting residue is dissolved in 20 ml of acetone followed bythe addition of 50 ml hexane. After standing in the refrigerator theproduct, so formed, is filtered and dried.

EXAMPLE 13 2-(1,2-Dimethyl-4-pyrrolyl)-4-(N-tosyl-L-alanyloxy)thiazol

5.0 g of 2-(1,2-dimethyl-4-pyrrolyl)-4-thiazol is dissolved in acosolvent of 100 ml of dichloromethane and 10 ml pyridine and cooled to5° C. A solution of 15.0 g of N-tosyl-L-alanyl chloride in 50 mldichloromethane is then added dropwise to the above mixture. Thereaction solution is stirred at room temperature or until the reactionis complete. The solution is washed with 1N hydrochloric acid, water,saturated sodium bicarbonate, and a saturated sodium chloride solutionin that order; dried over magnesium sulfonate; filtered; andconcentrated under reduced pressure. The residue, so formed, isdissolved in 100 ml of acteone and 100 ml of hexane is added. Afterstanding in the refrigerator for 1 hour, dark semisolid impurities aredecanted off and the remaining solvent evaporated. The amorphous solid,so formed, is dissolved in a minimum of ethylacetate, the resultingsolution is then applied to a silica column and eluted with a 2:1ethylacetate/hexane solvent system. Fractions containing the desiredproduct are collected, the solvent is evaporated, and the resultingresidue is dissolved in 20 ml of acetone followed by the addition of 50ml hexane. After standing in the refrigerator the product, so formed, isfiltered and dried.

EXAMPLE 14 2-(3-Indolyl)-4-(N-tosyl-L-alanyloxy)thiazol

5.0 g of 2-(3-indolyl)-4-thiazol is dissolved in a cosolvent of 100 mlof dichloromethane and 10 ml pyridine and cooled to 5° C. A solution of15.0 g of N-tosyl-L-alanyl chloride in 50 ml dichloromethane is thenadded dropwise to the above mixture. The reaction solution is stirred atroom temperature until the reaction is complete. The solution is washedwith 1N hydrochloric acid, water, saturated sodium bicarbonate, and asaturated sodium chloride solution in that order; dried over magnesiumsulfonate; filtered; and concentrated under reduced pressure. Theresidue, so formed, is dissolved in 100 ml of acteone and 100 ml ofhexane is added. After standing in the refrigerator for 1 hour, darksemisolid impurities are decanted off and the remaining solventevaporated. The amorphous solid, so formed, is dissolved in a minimum ofethylacetate, the resulting solution is then applied to a silica columnand eluted with a 2:1 ethylacetate/hexane solvent system. Fractionscontaining the desired product are collected, the solvent is evaporated,and the resulting residue is dissolved in 20 ml of acetone followed bythe addition of 50 ml hexane. After standing in the refrigerator theproduct, so formed, is filtered and dried.

EXAMPLE 15 2-(2-Furyl)-4-(N-tosyl-L-alanyloxy)thiazol

5.0 g of 2-(2-furyl)-4-thiazol is dissolved in a cosolvent of 100 ml ofdichloromethane and 10 ml pyridine and cooled to 5° C. A solution of15.0 g of N-tosyl-L-alanyl chloride in 50 ml dichloromethane is thenadded dropwise to the above mixture. The reaction solution is stirred atroom temperature until the reaction is complete. The solution is washedwith 1N hydrochloric acid, water, saturated sodium bicarbonate, and asaturated sodium chloride solution in that order; dried over magnesiumsulfonate; filtered; and concentrated under reduced pressure. Theresidue, so formed, is dissolved in 100 ml of acteone and 100 ml ofhexane is added. After standing in the refrigerator for 1 hour, darksemisolid impurities are decanted off and the remaining solventevaporated. The amorphous solid, so formed, is dissolved in a minimum ofethylacetate, the resulting solution is then applied to a silica columnand eluted with a 2:1 ethylacetate/hexane solvent system. Fractionscontaining the desired product are collected, the solvent is evaporated,and the resulting residue is dissolved in 20 ml of acetone followed bythe addition of 50 ml hexane. After standing in the refrigerator theproduct, so formed, is filtered and dried.

EXAMPLE 16 2-(β-Styryl)-4-(N-tosyl-L-alanyloxy)thiazol

5.0 g of 2-(β-styryl)-4-thiazol was dissolved in a cosolvent of 100 mlof dichloromethane and 10 ml pyridine and cooled to 5° C. A solution of15.0 g of N-tosyl-L-alanyl chloride in 50 ml dichloromethane was thenadded dropwise to the above mixture. The reaction solution was stirredat room temperature until the reaction was complete. The solution waswashed with 1N hydrochloric acid, water, saturated sodium bicarbonate,and a saturated sodium chloride solution in that order; dried overmagnesium sulfonate; filtered; and concentrated under reduced pressure.The residue, so formed, was dissolved in 100 ml of acteone and 100 ml ofhexane was added. After standing in the refrigerator for 1 hour, darksemisolid impurities were decanted off and the remaining solventevaporated. The amorphous solid, so formed, was dissolved in a minimumof ethylacetate, the resulting solution applied to a silica column andeluted with a 2:1 ethylacetate/hexane solvent system. Fractionscontaining the desired product were collected, the solvent wasevaporated, and the resulting residue was dissolved in 20 ml of acetonefollowed by the addition of 50 ml hexane. After standing in therefrigerator the product, so formed, was filtered and dried.

EXAMPLE 17 Preparation of a Leukocyte Diagnostic Device

To detect the presence of leukocytes in urine, a test strip containing acompound of the invention was prepared. A small type of testing paper ina regular square was attached to the end of a polystyrene strip,sedimented, and dried with the following two mixing solutionssuccessively. The first solution (100 ml of aqueous solution) contained5% (w/v) boric acid (pH 7.7) and 2% (w/v) polyvinyl pyrrolidone (K-10).The sedimented paper was dried by heating at 60° C. for 10 minutes. Asecond solution (100 ml of acetone) contained 0.06% (w/v) of2-(2-thienyl)-4-(N-tosyl-L-alanyloxy)thiazol or2-(β-styryl)-4-(N-tosyl-L-alanyloxy)thiazol; 0.05% (w/v)2-methoxy-4-morpholinobenzene diazonium chloride zinc chloride disaltsand 1.0% (w/v) n-decanol. The sedimented paper was dried by heating at50° C. for 5 minutes.

Successful leukocyte assays (i.e., the assays were useful for correctlyidentifying the presence of leukocytes in urine) were carried out usinga diagnostic device that contained each one of the thiazole esterslisted in this Example.

Each assay was conducted by contacting a test strip (manufactured asdescribed above) with a urine sample. When the urine sample containedleukocytes, the test strip showed the appearance of a violet color. Theinitial tint of violet color appeared within about 10 seconds andgradually darkened until the reaction was complete, which was about 1minute. The completed reaction showed a degree of color change that wassuitable not only for identifying whether leukocytes were present in theurine sample but also for semiquantitatively determining theconcentration of the leukocytes present.

EXAMPLE 18 Preparation of a Leukocyte Diagnostic Device

To detect the presence of leukocytes in urine, a test strip containing acomposition of the invention is prepared. A small type of testing paperin a regular square is attached to the end of a polystyrene strip,sedimented, and dried with the following two mixing solutionssuccessively. The first solution (100 ml of aqueous solution) contains5% (w/v) boric acid (pH 7.7) and 2% (w/v) polyvinyl pyrrolidone (K-10).The sedimented paper is dried by heating at 60° C. for 10 minutes. Asecond solution (100 ml of acetone) contains 0.06% (w/v) of2-(4-pyridyl)-4-(N-tosyl-L-alanyloxy)thiazol;2-(1-naphthyl)-4-(N-tosyl-L-alanyloxy)thiazole;2-(1,2-dimethyl-4-pyrrolyl)-4-(N-tosyl-L-alanyloxy)thiazol;2-(2-furyl)-4-(N-tosyl-L-alanyloxy)thiazol; or2-(3-indolyl)-4-(N-tosyl-L-alanyloxy)thiazol; 0.05% (w/v)2-methoxy-4-morpholinobenzene diazonium chloride zinc chloride disaltsand 1.0% (w/v) n-decanol. The sedimented paper is dried by heating at50° C. for 5 minutes.

Each assay is conducted by contacting a test strip (manufactured asdescribed above) with a urine sample. When the urine sample containsleukocytes, the test strip shows the appearance of a violet color. Theinitial tint of violet color appears within about 10 seconds andgradually darkens until the reaction is complete, which is about 1minute. The completed reaction shows a degree of color change that issuitable not only for identifying whether leukocytes are present in theurine sample but also for semiquantitatively determining theconcentration of the leukocytes.

The above specification, examples, and data provide a completedescription of the manufacture and the use of the many methods,compounds, compositions, and devices of the invention. Since manyembodiments of the invention can be made without departing from thespirit and the scope of the invention, the invention resides in theclaims hereinafter appended.

What is claimed is:
 1. A compound of the formula:

or a salt or solvated salt thereof, wherein A is an N-blocked amino acid residue or N-blocked peptide chain; and R₁ is unsubstituted or substituted heteroaryl; alkenyl substituted with unsubstituted or substituted aryl; or alkenyl substituted with unsubstituted or substituted heteroaryl.
 2. The compound of claim 1, wherein R₁ is heteroaryl substituted with alkyl, alkoxy, alkenyl, amino, acyl, halo, nitro, cyano, hydroxy, or —SO₃H.
 3. The compound of claim 1, wherein R₁ is unsubstituted or substituted heteroaryl selected from the group consisting of a 5-membered ring and a 6-membered ring.
 4. The compound of claim 1, wherein R₁ is unsubstituted or substituted heteroaryl selected from the group consisting of a nitrogen-containing ring, an oxygen-containing ring, and a sulfur-containing ring.
 5. The compound of claim 1, wherein R₁ is alkenyl selected from the group consisting of ethenyl, propenyl, and butenyl, the alkenyl being substituted with unsubstituted or substituted aryl or unsubstituted or substituted heteroaryl.
 6. The compound of claim 1, wherein R₁ is alkenyl substituted with aryl that is unsubstituted phenyl or substituted phenyl.
 7. The compound of claim 6, wherein the phenyl is substituted with alkyl, alkenyl, alkoxy, amino, acyl, halo, nitro, cyano, hydroxy, or —SO₃H.
 8. The compound of claim 1, wherein R₁ is alkenyl substituted with substituted heteroaryl, wherein the heteroaryl is substituted with alkyl, alkoxy, alkenyl, amino, acyl, halo, nitro, cyano, hydroxy, or —SO₃H.
 9. The compound of claim 1, wherein R₁ is alkenyl substituted with heteroaryl that is selected from the group consisting of a 5-membered ring and a 6-membered ring.
 10. The compound of claim 1, wherein R₁ is alkenyl substituted with heteroaryl that is selected from the group consisting of a nitrogen-containing ring, an oxygen-containing ring, and a sulfur-containing ring.
 11. The compound of claim 1, wherein R₁ is heteroaryl substituted with alkyl or alkoxy.
 12. The compound of claim 1, wherein the compound is of the formula:


13. The compound of claim 1, wherein the compound is of the formula:


14. The compound of claim 1, wherein the compound is of the formula:


15. The compound of claim 1, wherein the compound is of the formula:


16. The compound of claim 1, wherein the compound is of the formula:


17. The compound of claim 1, wherein the compound is of the formula:


18. The compound of claim 1, wherein A is N-blocked alanine or N-blocked polyalanine.
 19. A compound of the formula:

or a salt or solvated salt thereof, wherein A is an N-blocked amino acid residue or N-blocked peptide chain; and R₂ is fused hydrocarbyl rings in which at least one ring is aromatic, and there is at least one substituent on said fused hydrocarbyl rings.
 20. The compound of claim 19, wherein R₂ is selected from the group consisting of naphthyl, tetrahydronaphthyl, and anthryl.
 21. The compound of claim 19, wherein the fused hydrocarbyl rings are substituted on at least one ring with alkyl, alkoxy, alkenyl, amino, acyl, halo, nitro, cyano, hydroxy, or —SO₃H.
 22. The compound of claim 19, wherein the fused hydrocarbyl rings contain at least one ring selected from the group consisting of a 5-membered ring and a 6-membered ring.
 23. The compound of claim 19, wherein the fused hydrocarbyl rings are substituted on at least one ring with alkyl or alkoxy.
 24. The compound of claim 19, wherein the compound is of the formula:


25. The compound of claim 19, wherein A is N-blocked alanine or N-blocked polyalanine.
 26. A composition for detecting leukocytes comprising: (a) a compound of the formula:

 or a salt or solvated salt thereof, wherein A is an N-blocked amino acid residue or N-blocked peptide chain, and R₁ is unsubstituted or substituted heteroaryl; alkenyl substituted with unsubstituted or substituted aryl; or alkenyl substituted with unsubstituted or substituted heteroaryl; and (b) a diazonium salt.
 27. The composition of claim 26, wherein the compound has R₁ that is heteroaryl substituted with alkyl, alkoxy, alkenyl, amino, acyl, halo, nitro, cyano, hydroxy, or —SO₃H.
 28. The composition of claim 26, wherein the compound has R₁ that is unsubstituted or substituted heteroaryl that is selected from the group consisting of a 5-membered ring and a 6-membered ring.
 29. The composition of claim 26, wherein the compound has R₁ that is unsubstituted or substituted heteroaryl selected from the group consisting of a nitrogen-containing ring, an oxygen-containing ring, and a sulfur-containing ring.
 30. The composition of claim 26, wherein the compound has R₁ that is alkenyl selected from the group consisting of ethenyl, propenyl, and butenyl, the alkenyl being substituted with unsubstituted or substituted aryl or unsubstituted or substituted heteroaryl.
 31. The composition of claim 26, wherein the compound has R₁ that is alkenyl substituted with aryl that is unsubstituted phenyl or substituted phenyl.
 32. The composition of claim 31, wherein the phenyl is substituted with alkyl, alkenyl, alkoxy, amino, acyl, halo, nitro, cyano, hydroxy, or —SO₃H.
 33. The composition of claim 26, wherein the compound has R₁ that is alkenyl substituted with heteroaryl, wherein the heteroaryl is substituted with alkyl, alkoxy, alkenyl, amino, acyl, halo, nitro, cyano, hydroxy, or —SO₃H.
 34. The composition of claim 26, wherein the compound has R₁ that is alkenyl substituted with heteroaryl selected from the group consisting of a 5-membered ring and a 6-membered ring.
 35. The composition of claim 26, wherein the compound has R₁ that is alkenyl substituted with heteroaryl that is selected from the group consisting of a nitrogen-containing ring, an oxygen-containing ring, and a sulfur-containing ring.
 36. The composition of claim 26, wherein the compound has R₁ that is heteroaryl substituted with alkyl or alkoxy.
 37. The composition of claim 26, wherein the compound is of the formula:


38. The composition of claim 26, wherein the compound is of the formula:


39. The composition of claim 26, wherein the compound is of the formula:


40. The composition of claim 26, wherein the compound is of the formula:


41. The composition of claim 26, wherein the compound is of the formula:


42. The composition of claim 26, wherein the compound is of the formula:


43. The composition of claim 26, wherein the compound has A that is N-blocked alanine or N-blocked polyalanine.
 44. The composition of claim 26, wherein the diazonium salt is selected from the group consisting of 1-diazo-8-naphtol-3,6-disulfonic acid, chloride, zinc chloride double salt; 6-diazo-1-naphtol-3-sulfonic acid, chloride double salt; and 2-methoxy-4-morpholinobenzene diazonium chloride, zinc chloride double salt.
 45. The composition of claim 26, wherein the composition is free of accelerating salt.
 46. A composition for detecting leukocytes comprising: (a) a compound of the formula

 or a salt or solvated salt thereof, wherein A is an N-blocked amino acid residue or N-blocked peptide chain; and R₂ fused hydrocarbyl rings in which at least one ring is aromatic and there is at least one substituent on said fused hydrocarbyl rings; and (b) a diazonium salt.
 47. The composition of claim 46, wherein the compound has R₂ selected from the group consisting of naphthyl, tetrahydronaphthyl, and anthryl.
 48. The composition of claim 46, wherein the fused hydrocarbyl rings are substituted on at least one ring with alkyl, alkoxy, alkenyl, amino, acyl, halo, nitro, cyano, hydroxy, or —SO₃H.
 49. The composition of claim 46, wherein the fused hydrocarbyl rings contain at least one ring selected from the group consisting of a 5-membered ring and a 6-membered ring.
 50. The composition of claim 46, wherein the fused hydrocarbyl rings are substituted on at least one ring with alkyl or alkoxy.
 51. The composition of claim 46, wherein the compound is of the formula:


52. The composition of claim 46, wherein the compound has A that is N-blocked alanine or N-blocked polyalanine.
 53. The composition of claim 46, wherein the diazonium salt is selected from the group consisting of 1-diazo-8-naphtol-3,6-disulfonic acid, chloride, zinc chloride double salt; 6-diazo-1-naphtol-3-sulfonic acid, chloride double salt; and 2-methoxy-4-morpholinobenzene diazonium chloride, zinc chloride double salt.
 54. The composition of claim 46, wherein the composition is free of accelerating salt.
 55. A compound of the formula:


56. A compound of the formula:


57. A compound of the formula:


58. A compound of the formula:


59. A compound of the formula:


60. A compound of the formula: 